While the 550-kDa PRK/GAPDH/ CP12 complex is dissociated and activated upon reduction alone, activation and dissociation of the 600-kDa A8B8 complex of NADP-GAPDH requires incubation wit
Trang 1Co-existence of two regulatory NADP-glyceraldehyde 3-P
dehydrogenase complexes in higher plant chloroplasts
Renate Scheibe1, Norbert Wedel2, Susanne Vetter1, Vera Emmerlich3and Sonja-Manuela Sauermann1 1
Plant Physiology, University of Osnabrueck, Germany;2Planton GmbH, Kiel, Germany;3Plant Physiology, University of
Kaiserslautern, Kaiserslautern, Germany
Light/dark modulation of the higher plant Calvin-cycle
enzymes phosphoribulokinase (PRK) and
NADP-depend-ent glyceraldehyde 3-phosphate dehydrogenase
(NADP-GAPDH-A2B2) involves changes of their aggregation state
in addition to redox changes of regulatory cysteines Here we
demonstrate that plants possess two different complexes
containing the inactive forms (a) of NADP-GAPDH and
PRK and (b) of only NADP-GAPDH, respectively, in
darkened chloroplasts While the 550-kDa PRK/GAPDH/
CP12 complex is dissociated and activated upon reduction
alone, activation and dissociation of the 600-kDa A8B8
complex of NADP-GAPDH requires incubation with
dithiothreitol and the effector 1,3-bisphosphoglycerate In
the light, PRK is therefore completely in its activated state
under all conditions, even in low light, while GAPDH
acti-vation in the light is characterized by a two-step mechanism
with 60–70% activation under most conditions in the light, and the activation of the remaining 30–40% occurring only when 1,3-bisphosphoglycerate levels are strongly increasing
In vitrostudies with the purified components and coprecipi-tation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates Iso-lated oxidized PRK alone does not reaggregate after it has been purified in its reduced form; only in the presence of both CP12 and purified NADP-GAPDH, some of the PRK reaggregates Recombinant GapA/GapB constructs form the A8B8complex immediately upon expression in E coli Keywords: enzyme aggregation; light/dark regulation; NADP-glyceraldehyde 3-P dehydrogenase; phospho-ribulokinase; spinach chloroplast
Various chloroplast enzymes are subjected to light/dark
modulation of their activity, brought about by a
redox-modification at specific cysteine residues mediated by the
ferredoxin/thioredoxin system [1] The activity of each of
these enzymes is adjusted by fine-tuning, the rates of
reduction and/or of oxidation being influenced by specific
metabolites [2] At constant redox conditions, this allows for
independent changes in the steady-state activities of each of
the enzymes merely by changes in the metabolic state of the
chloroplast [3,4] In some cases, the reversible changes of
redox and activation states are accompanied by
oligomeri-zation and re-dissociation of transient complexes
Enzyme aggregations of various compositions have been
described repeatedly, their occurrence under in vivo
condi-tions still being under debate [5,6] But even the actual
composition of enzyme aggregates containing
NAD(P)-dependent glyceraldehyde-3 P dehydrogenase
[NAD(P)-GAPDH] and phosphoribulokinase (PRK) is controversial
Both activities appear to occur in high-molecular-mass
forms in darkened chloroplasts, either in homo-oligomers
[7–10] or in hetero-oligomers [11,12], the latter involving a small chloroplast protein, CP12, with a high sequence similarity to the C-terminus of subunit B of the unique chloroplast form of GAPDH [13] Here we describe the presence of both types of aggregations in darkened spinach chloroplasts, one consisting of GAPDH A and B alone, the other consisting of PRK, GAPDH, and CP12 The differential stability of both complexes upon reductive activation, the formation of the A8B8-GAPDH complex from the recombinant subunits in Escherichia coli, the reconstitution of the hetero-oligomeric PRK/GAPDH/ CP12 complex from the isolated components in vitro, and coprecipitation from oxidized stroma using antisera against the individual components, support this fact
M A T E R I A L S A N D M E T H O D S
Isolation of intact chloroplasts and preparation
of concentrated stroma Darkened spinach leaves from plants in hydroponic culture were cut and homogenized in isotonic medium consisting of
330 mM mannitol, 30 mM Mops, 2 mM EDTA, pH 7.8, according to [14] The chloroplast pellet was washed twice and then treated on ice with a glass homogenizer using as little additional medium as possible All steps were per-formed in darkness After centrifugation, the cleared supernatant was filtered through a 0.2-lm filter The protein content was determined according to [15] with BSA as a standard The protein concentration was adjusted to 10 mg per 2 mL sample with column buffer (see gel filtration)
Correspondence to R Scheibe, Plant Physiology, University of
Osnabrueck, Osnabrueck D-49069, Germany.
Abbreviations: GapA/GapB, subunits A and B of GAPDH; GAPDH,
glyceraldehyde 3-P dehydrogenase; GSH and GSSG, reduced and
oxidized glutathione; PRK, phosphoribulokinase.
Enzymes: NAD(P)-dependent glyceraldehyde 3-phosphate
dehydrogenase (EC 1.2.1.13); phosphoribulokinase (EC 2.7.1.19).
(Received 3 June 2002, revised 21 August 2002,
accepted 18 September 2002)
Trang 2either without (dark) or with 20 mM dithiothreitol, and
incubated in a darkened vial for 30 min at 25C For
light-activation experiments, intact chloroplasts were isolated and
incubated in bicarbonate-containing medium as described
in [9]
Gel filtration on Superdex S-200
Preincubated samples were filtered through a calibrated
Superdex 200 column (Hiload 16/60) (Pharmacia, Freiburg,
Germany) at 1 mLÆmin)1, collecting fractions of 1 mL
which were used for enzyme assays The column buffer
consisted of 10 mM bicine/KOH, 150 mM NaCl, 140 lM
NAD, pH 7.8, for dark samples, and additionally 2.5 mM
dithiothreitol for dithiothreitol samples
Enzyme assays
Aliquot samples of the fractions were either assayed directly
or after preincubation with 50 mMdithiothreitol (for PRK
activation) or with 20 mM dithiothreitol and 21 lM
1,3-bisphosphoglycerate in a regenerating system as in [16] for
GAPDH activation The 1,3-bisphosphoglycerate-system as
fivefold stock solution contained 100 mMTris/HCl, pH 7.8,
8 mMMgSO4, 1 mMEDTA, 4 mMATP, 9 mM3-PGA and
PGK (3.6 UÆmL)1) The PRK assay was as in [17]
NADP-dependent GAPDH activity was determined as in [16]
Protein purification
PRK was purified from spinach leaves according to [18]
with some modifications During all purification steps
10 mMdithiothreitol was present The proteins precipitating
between 40 and 55% of the saturation of ammonium sulfate
at 4C were resuspended and subjected to acid precipitation
with acetic acid at pH 5.0 The supernatant was adjusted to
pH 6.8 and dialyzed over night The diluted and clarified
solution in 10 mM bicine/KOH, 10 mM dithiothreitol,
pH 6.8, was subjected to affinity chromatography on
Reactive Red (Merck, Darmstadt, Germany), washed with
the same buffer After a washing step with 10 mMpotassium
phosphate, 10 mM dithiothreitol, pH 6.9, the PRK was
eluted in 10 mM potassium phosphate, pH 7.2, 10 mM
dithiothreitol, and 5 mM ATP The fractions with PRK
activity were concentrated by ammonium sulfate
precipita-tion (80% of saturaprecipita-tion) and resuspended in 50 mMbicine/
KOH, 10 mMpotassium phosphate, 1 mMEDTA, 10 mM
dithiothreitol, 10% glycerol, pH 8.0, and subjected to gel
filtration in 100 mM bicine/KOH, 10 mM dithiothreitol,
1 mMEDTA, pH 8.0 Storage of the active dimeric protein
was in 50% glycerol at)20 C
GADPH was purified from spinach leaves as described
by [19], slightly modified as in [16] GapA and GapB were
expressed as a combined construct in E coli BL21DE3
pLys S containing the two clones each unter the control of
the T7 promoter, and the kanamycin and the ampicillin
resistance gene, respectively, in order to control the presence
of both constructs
CP12 from spinach was produced in E coli with an
N-terminal His-tag and was purified using Ni-chelating
chromatography Elution was achieved with 1Mimidazol,
0.5MNaCl in 20 mMTris/HCl, pH 7.9 For reconstitution
experiments the His-tag was removed by proteolysis using
thrombin according to the manufacturer’s protocol (Strate-gene, Heidelberg, Germany) The CP12 clone for the mature protein was originally described in [13]
Artificial stroma conditions Reconstitution experiments with the purified proteins were performed in a solution simulating the high protein conditions in the stromal sample using a modified artificial stroma [20] In more detail, the sample for dark conditions consisted of 5 mM MgSO4, 10 mM NaCl, 5 mM KNO3,
5 mM KH2PO4, 25 mM sucrose, 20 mM glucose, 5 mM fructose, 40 lMNAD, 20 mMGSSG, the purified proteins (each about 200 lg) and BSA (defatted) to reach a total protein content of 10 mg per 2 mL sample Incubation was
30 min at room temperature and then at 4C over night The purified GAPDH was pretreated with 100 lM 1,3-bisphosphoglycerate in a regenerating system and desalted
in order to obtain the oxidized A2B2 form prior to the reconstitution assay
SDS/PAGE, Western blotting and immunodecoration Equal volumes of fractions after gel filtration were subjected
to SDS/PAGE (12% acrylamide), and the gels were electroblotted and immunodecorated as in [12]
Immunoprecipitation Protein-A Sepharose was preincubated with either preim-mune serum or the indicated antisera in Tris-buffered saline for 30 min at room temperature, washed once and then added to stromal extracts that had been preincubated with
140 lM NAD and 20 mM GSSG After incubation for
30 min, the supernatant was used to assay for GAPDH and PRK activities after full activation The antisera against CP12 and NADP-GAPDH have been obtained from rabbits using the spinach proteins purified from E coli and spinach, respectively Antiserum against PRK was a kind gift from Fred Hartman, Oak Ridge, USA
R E S U L T S
Reversible aggregation of GAPDH and PRK
in chloroplasts When the soluble fraction of isolated darkened spinach chloroplasts was subjected to gel filtration, both GAPDH and PRK were obtained as high-molecular-mass aggregates (Fig 1A) Enzyme activities in the fractions were detected after full activation The activity peaks do not coincide completely, GAPDH eluting somewhat earlier than PRK in all cases This tendency was even more pronounced in maize chloroplasts (Fig 2), where the GAPDH activity formed a distinct shoulder at 600 kDa in addition to the peak at
550 kDa that coincided with the PRK activity The high-molecular-mass form of GAPDH is almost inactive due to its decreased affinity for 1,3-bisphosphoglycerate [16] PRK
in darkened chloroplasts when eluted as the 550-kDa form
is also inactive; its activity is only retrieved upon preincu-bation of the fractions with dithiothreitol (Fig 3A) On the other hand, the dimeric PRK as obtained from illumin-ated chloroplasts is eluted as fully active enzyme (Fig 3B)
Trang 3PRK purified in its reduced dimeric state [18] can be
reversibly inactivated by treatment with oxidant [21], but
remains dimeric (Fig 3C)
Preincubation of spinach chloroplasts with 20 mM
dithio-threitol for 30 min resulted in the complete disappearance
of high-molecular-mass PRK and in the partial ( 50%)
dissociation of the GAPDH aggregates (Fig 1B) From
experiments with the purified enzyme it is known that
reductive treatment of the 600-kDa A8B8-GAPDH form
does not lead to any increase in activity nor to dissociation
[16] From the two-peak elution pattern of the GAPDH
activity, we therefore assumed the presence of two types of
GAPDH-containing aggregates, namely the A8B8complex
which was still intact after incubation with dithiothreitol
alone [16], and the GADPH/PRK/CP12 complex described
by [12] which, upon reduction, releases GAPDH and PRK
as tetramer and dimer, respectively CP12 and PRK could
not be detected by the respective antisera in the high-molecular-mass fractions after dithiothreitol treatment, while all three proteins were detectable with antisera in the peak fractions of the untreated sample (Fig 1C,D) The high-molecular-mass fraction did not contain other enzyme activities such as phosphoglycerate kinase [22] or fructose 1,6-bisphosphatase [23] that had been suggested to also form high-molecular-mass aggregates (data not shown) Furthermore, we never detected any tetramic GAPDH in dark stroma which has been suggested to occur as A4by [24]
Differential activation behaviour for GAPDH and PRK upon dithiothreitol and light treatment
Incubation of chloroplast stroma with increasing concen-trations of dithiothreitol resulted in 100% activation of PRK even at low dithiothreitol concentrations at pH 8.0 (Fig 4A) In contrast, even up to 20 mMresulted in only 60–70% for the maximal GAPDH activity Only in the presence of added ATP and/or 3-phosphoglycerate, thus increasing the 1,3-bisphosphoglycerate concentration in the stroma [9], 100% activation of GAPDH was reached This
is true also for activation by light, where 100% of PRK and 40–60% of GAPDH activity were reached already at very low light intensities These levels were unchanged over a wide range of light intensities, only addition of ATP to the isolated chloroplasts increased the level of GAPDH activa-tion to 100% (Fig 4B) This is in agreement with the fact that both reduction and 1,3-bisphosphoglycerate are required for activation and dissociation of GAPDH from the A8B8form [9,16]
Reaggregation of PRK and GAPDH from chloroplast fractions
Purification of PRK according to published procedures [3,17] is always performed in the presence of dithiothreitol, leading to the preparation of the enzyme in its reduced active form Using this enzyme, the mechanism of reversible
Fig 1 Gel filtration of spinach chloroplast
stroma from darkened leaves The extract was
preincubated either in the absence (A, C) or in
the presence of 10 m M dithiothreitol (DTT)
(B, D) for 30 min at 25 C The activities of
PRK (s) and of NADP-GAPDH (d) were
determined in aliquot fractions after full
activation as described under Materials and
methods Every second fraction was subjected
to SDS/PAGE The gel was stained with
Poinceau Red (0.2% in 3% (w/v)
trichloro-acetic acid), destained, and immunodecorated
with the indicated antisera (C,D) Fractions
reacting with the antiserum against CP12 are
highlighted with an arrow.
Fig 2 Gel filtration of chloroplast stroma from darkened maize leaves
in the absence of dithiothreitol PRK (s) and NADP-GAPDH (d)
activities were determined after full activation as described in Materials
and methods.
Trang 4redox-modification mediated by thioredoxin has been
analyzed in much detail The redox-active Cys residues
have been identified (Cys15 and Cys55) [25], their redox
potential has been determined [3,26], and the interaction
with thioredoxin f has been studied [21]
In order to analyze the structural changes upon redox
modification, we have reoxidized the purified, reduced
enzyme Incubation with 50 mM dithiothreitol or with
25 mM GSSG at pH 8.0 resulted in an almost complete inactivation This inactivation could be reversed by the addition of reductant (Table 1) However, both the reduced and the oxidized enzyme forms appeared as dimers upon gel filtration (Table 1) (Fig 3C) This is in contrast to the experiments with chloroplast stroma, where the oxidized
Fig 3 Gel filtration of spinach chloroplast stroma obtained from
darkened (A)or from illuminated (B)organelles The PRK activity in
the fractions was either determined directly (s) or after full activation
(d) In (C), purified enzyme either in its original reduced state (d) or
after oxidation with GSSG (s) was subjected to gel filtration.
Fig 4 Activities of PRK and NADP-GAPDH: (A)dependence upon dithiothreitol concentration in a stromal extract and (B)dependence on light intensity in intact chloroplasts (A) The stromal extract was pre-incubated with the given concentrations of dithiothreitol for 30 min at
25 C (d,s) A parallel sample was incubated with dithiothreitol (DTT) at the various concentrations and 21 l M 1,3-bisphosphogly-cerate-regenerating system (m) The samples were assayed for PRK (s) and NADP-GAPDH activity (d,m) (B) The intact chloroplasts were incubated in the presence of 5 m M sodium bicarbonate (d,s) and
in addition 0.5 m M 3-phosphoglycerate and 0.25 m M ATP (m).
Table 1 Activity and aggregation state of purified spinach PRK Reduced PRK was purified in the presence of 10 m M dithiothreitol Oxidized PRK was treated with 25 m M GSSG or with 50 m M oxidized dithiothreitol, pH 8.0, for 15 min at 20 C.
Reduced Oxidized Activity (UÆmg protein)1) 437 8 Molecular mass (kDa) 80 80
Trang 5dark form of PRK appears as high-molecular-mass form.
Such controversial behaviour has been described already
[8,21]
In order to investigate the requirement for a small stromal
protein (i.e CP12) for reaggregation of dimeric PRK, we
separated the dissociated enzyme (Fraction II: 80–120 kDa
in Fig 5A) from the higher-molecular-mass fraction at
600 kDa (Fraction I) by gel filtration Then a concentrated
fraction containing the smaller stromal proteins (Fraction
III: 20–60 kDa) and the enzyme fraction II were incubated
together with GSSG After another step of gel filtration, a
new high-molecular-mass fraction became apparent
con-taining PRK activity (after reductive activation of the
fractions) (Fig 5B) This peak contained almost equal
activities of GAPDH and PRK
Reconstitution of GAPDH (A8B8) and PRK/GAPDH/CP12
complexes
In a further approach, we attempted to reconstitute both
complexes from the purified components For these
experi-ments, the purified proteins were kept in an artificial stroma according to [20] containing various ions, sugars, and amino acids (see Materials and methods) and in addition 140 lM NAD, 10 mM GSSG and BSA (20 mgÆmL)1) in order to simulate the conditions of high protein concentration in the chloroplast stroma Under these conditions, absolutely no aggregation was observed with PRK and CP12 alone (Fig 6A) In contrast, incubation of PRK with purified GAPDH and CP12 resulted in the aggregation of some of
Fig 5 Gel filtration of spinach chloroplast stroma in the presence of
dithiothreitol and reaggregation of the components of the GAPDH/
PRK/CP12 complex After gel filtration on Superdex 200 of
dithio-threitol-treated stroma, the combined fractions II and III as indicated
in (A) were concentrated and incubated over night (B) After another
passage through Superdex 200, the fractions were assayed for PRK (s)
and NADP-GAPDH (d) activity after full activation.
Fig 6 Reconstitution of PRK/GAPDH/CP12 and GAPDH (A 8 B 8 ) complex from the purified/recombinant components (A, B) The purified proteins were incubated in artificial stroma as described under Materials and methods GAPDH (400 lg) pretreated with 380 l M
1,3-bisphosphoglycerate and desalted in order to generate the tetra-meric A 2 B 2 form, was present in addition to 200 lg purified PRK and
200 lg recombinant CP12 (C) Recombinant GAPDH consisting of the subunits A and B in the soluble extract from IPTG-induced E coli cells was directly applied to a Superdex-200 gel filtration column All proteins were kept in their oxidized form by incubation with 20 m M
GSSG.
Trang 6the PRK and all of the GAPDH (Fig 6B) The low yield of
PRK reaggregation was probably due to the rather artificial
conditions Some of the aggregated GAPDH was most
likely in its A8B8form
Finally, the expression of both GAPDH subunits in
E coli was performed A high-molecular-mass complex
formed immediately in E coli upon expression of the
combined construct for GapA and GapB and eluted as a
600-kDa form (Fig 6C) The activation characteristics of
the recombinant GAPDH are typical for the A8B8form,
which is still present in the 600-kDa peak in
dithiothreitol-treated stroma (fraction I) In both cases, activation was
obtained only after incubation with both dithiothreitol and
1,3-bisphosphoglycerate, not with dithiothreitol alone
(Fig 7)
Immunoprecipitation
Using the specific antisera against the components of the
two complexes, namely GAPDH, PRK and CP12,
GSSG-oxidized stromal extracts were fractionated In order not to
disturb the existing complexes, the immunoglobulin fraction
was bound to immobilized Protein A and washed with
50 mM bicine/KOH, pH 8.0, before it was exposed to
oxidized stroma containing 140 lM NAD which stabilizes
the complexes and also the enzyme activities The enzyme
composition in the supernatant was quantified from the
activities obtained after complete activation The results are
shown in Table 2 With antiserum against NADP-GAPDH
about 82.5% of the PRK activity could be coprecipitated
with 91.5% of the GAPDH activity, indicating that only
17.5% of the PRK was not associated with GAPDH in a
complex On the other hand, the antiserum against PRK
removed only 42.9% of the GAPDH from the solution, together with 96.1% of the PRK This again indicates the presence of an independent GAPDH-A8B8complex apart from the PRK/GAPDH/CP12-mixed complex The fact that the antiserum raised against recombinant CP12 only removed a small proportion of the enzymes from the solution, is most likely due to inaccessibility of the CP12 in the native complex, because the serum recognized native soluble CP12 in reduced stromal extracts (data not shown)
D I S C U S S I O N
In general, evidence is increasing that cellular contents are well organized in microcompartments due to protein– protein interactions between partners of a metabolic path-way or of a signal transduction cascade In particular for chloroplasts, there have been various attempts to show the presence of bi- or multi-enzyme complexes of Calvin-cycle enzymes (reviewed in [27]); however, there are intrinsic technical problems when trying to confirm any of the interactions unambiguously The critical step is always the breakage of the cell or the organelle, since changes of the protein concentration and of the low-molecular-mass components of the soluble medium will occur
In order to avoid any new formation of complexes, ammonium sulfate precipitation has been omitted in our procedure and stromal fractions were applied directly to the gel filtration column This leads to reproducable elution profiles on the Superdex 200 column in the presence of
150 mMNaCl, with two distinguishable peaks at 600 and
550 kDa eluting well after the void volume It should be pointed out, however, that the protein concentration of the stroma sample for gel filtration is critical to obtain the described results [28]
Rubisco is eluted at the same position as PRK (550 kDa), but the subunit composition (L8S8) of the former suggests that further enzymes are not associated in the same complex, although this assumption is in contrast to the results of Rault et al [6] who purified a five-enzyme complex of 540 kDa containing Rubisco, GAPDH, PRK, phosphoriboseisomerase, and 3-phosphoglycerate kinase Other activities, such as FBPase that had been described to
be subject to aggregation [23] or had been seen as part of the so-called photosynthetosome [5] could not be detected in this range It cannot be excluded, however, that weaker interactions are involved in such supramolecular organiza-tion observed by other groups that are therefore not stable under the conditions applied in this study
Previously we had identified and characterized the A8B8 -GAPDH form present in darkened chloroplasts [9,16] Later, we showed by limited proteolysis [29] and using truncated constructs expressed in E coli [30] that the unique C-terminal sequence extension of GapB is responsible for
Fig 7 Activation properties of A 8 B 8 -GAPDH in stromal extract and in
E coli GAPDH activites in the 600-kDa peak fractions of
dithio-threitol-treated stroma and of the recombinant GapA/GapB
prepar-ation were determined The pooled fractions of peak I from Fig 5A
(grey bars) and from Fig 6C (hatched bars) were either assayed
directly (– DTT), after incubation with 10 m M dithiothreitol
(+ DTT), or with 10 m M dithiothreitol and 21 lg
1,3-bisphospho-glycerate (+ DTT/1,3bisPGA) DTT, dithiothreitol.
Table 2 Percentage coprecipitation of PRK-, GAPDH- and CP12-containing complexes from oxidized stroma The activities remaining in the supernatant were determined after full activation.
Antiserum NADP-GAPDH PRK
Trang 7aggregation and inactivation of GAPDH in the dark.
Likewise, expression of the complete subunit B in E coli
had led to aggregated forms [30,31] Dissociation and
activation of the A8B8complex requires
1,3-bisphosphogly-cerate [9,16,32]
Analyzing PRK activation, there was an obvious
dis-crepancy between in vitro results with the purified enzyme
and results obtained after gel filtration of chloroplast
stroma, a fact also observed by [21] Therefore, it was
assumed that PRK had lost a component enabling
aggre-gation in vivo, and re-addition of a low-molecular-mass
stromal fraction to the fraction of dissociated enzymes
indeed allowed reaggregation (see Fig 5)
In this paper, we have attempted to characterize such a
PRK-containing complex, having in mind the postulated
existence of a mixed GAPDH–PRK complex containing
CP12 as described previously [33] Therefore it was
neces-sary to separate the A8B8form of GAPDH and such mixed
complex This could be achieved by various means due to
their diverging properties upon activation and dissociation
In addition, reconstitution and coprecipitation experiments
using specific antisera helped to confirm the presence of two
different complexes Unfortunately, the anti-CP12 serum
was not able to reach this protein when part of the complex
due to sterical hindrance Taken together, it could be
established that GAPDH activity is present in two different
complexes reacting with distinct sensibility towards
dithio-threitol and the effector 1,3-bisphosphoglycerate leading to
dissociation and activation On the other hand, all PRK
activity was present in a mixed complex forming only in the
presence of both GAPDH and CP12
In order to understand the differential regulation of
Calvin-cycle enzymes, their midpoint redox potentials have
been determined and compared to physiological data
obtained with the intact system [34] The apparent
discrep-ancies with respect to GAPDH could be easily explained
with the presence of two differently responding
GAPDH-containing aggregates: (a) the early evolved system, namely
PRK/GAPDH/CP12, which is activated merely by
reduc-tion and is already present in cyanobacteria [33]; and (b) the
A8B8form that evolved with the appearance of multicellular
green organisms when GapB emerged in Characeae for the
first time (R Cerff and J Peterson, Institut fu¨r Genetik, Tu
Braunschweig, Germany, personal communication)
The occurrence of both aggregates in plants indicates their
special role for optimal photosynthesis under all conditions
A fast-responding system for complete PRK activation and
activation of a portion of GAPDH is required for CO2
assimilation under rather constant conditions, and is thus
available in all photosynthetic organisms Green plants with
less constant environments (evolution of land plants)
acquired in addition a larger fraction of GAPDH activity
that, however, remains latent until a certain level of the
substrate 1,3-bisphosphoglycerate indicates the demand to
increase the flux specifically at this point For the situation in
higher plants this means that there is a two-level activation of
GAPDH, reaching 50–60% of the total activity even under
low light and low reductant concentrations and 100%
activation only with at increased 1,3-bisphosphoglycerate
levels Full activation of PRK is achieved under all
conditions (see Fig 4) Calculating the stoichiometry of
the enzyme subunits engaged in the two complexes using the
specific activities of the purified enzymes (GAPDH 200,
PRK 400 UÆmg protein)1) and the activities present in the stroma (700 and 300 lmolÆmg Chl)1Æh)1) The ratio of 2 GAPDH/PRK/CP12 complexes to 1 A8B8 complex sup-ports the finding of a ratio of 1 : 1 of the total GAPDH activity in both complexes
From our experience, even very dim light during chloro-plast isolation or preparation of extract leads to a significant level of PRK activity and to some basic GAPDH activity that is often described as dark activity In contrast, strict darkness during these steps will result in complete inactiva-tion The residual GAPDH activity obtained in vitro is rather the result of the high 1,3-bisphosphoglycerate con-centration (21 lM) in the standard assay leading to some activity of the inactivated enzyme (low affinity for substrate 1,3-bisphosphoglycerate) as opposed to the in vivo situation with a stromal concentration of 1–2 lM 1,3-bisphospho-glycerate [9,16]
Regulation of the activity at the PRK step is exclusively achieved by noncovalently acting inhibitors, with ribulose 1,5-P2 (Ki¼ 0.7 mM), 3-phosphoglycerate (Ki¼ 2 mM), inorganic phosphate (Ki¼ 4 mM) and ADP (Ki¼ 40 lM),
to list only some of them [35], since even mild reduction results in complete dissociation of PRK from the GAPDH/ PRK/CP12 complex and thus PRK activation
A C K N O W L E D G E M E N T S
The authors thank Dr Carsten Sanders, and Dr Simone Holtgrefe, Osnabru¨ck, for initial experiments in the preparation of the clones and some chloroplast experiments Thanks are also due to Dipl.-Biol Elisabeth Baalmann and Dr Jan E Backhausen, Osnabru¨ck, for helpful discussion and to Prof Dr R Cerff, Braunschweig, for initial encouragement and advice Finally, the financial support given by the Deutsche Forschungsgemeinschaft to Renate Scheibe (Sche 217/8) is gratefully acknowledged.
R E F E R E N C E S
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